Cellular protein quality control systems enable the robust response to protein misfolding that is essential for normal cellular function. Numerous pathologies including neurodegenerative disorders, ataxias, and cancers can result from defects in the protein quality control system formed by the interaction of the E3 ubiquitin ligase CHIP (C-terminus of Hsp70 Interacting Protein) and the ATP-dependent chaperone Hsp70 (70 kilodalton heat shock protein). Despite the importance of the CHIP/Hsp70 protein quality control complex, the triage mechanism used by the CHIP/Hsp70 complex to target misfolded proteins for either Hsp70-mediated refolding or ubiquitin proteasome-mediated degradation is unknown. Within the triage mechanism, several key areas remain unexplored. These include: (1) How does the CHIP/Hsp70 complex execute the observed triage mechanism that ubiquitinates misfolded clients first, then Hsp70, then CHIP? (2) What is the role played by nucleotide occupancy of Hsp70 and how do the conformational changes in Hsp70 throughout the catalytic cycle dictate the ability of CHIP to ubiquitinate misfolded clients? (3) What are the determinants that control whether a misfolded client becomes ubiquitinated, or is refolded? The proposed research program will examine these issues using nuclear magnetic resonance (NMR), small angle X-ray scattering (SAXS), electron paramagnetic resonance (EPR), and a suite of biophysical and biochemical techniques. NMR data will provide high-resolution information on dynamics within CHIP/Hsp70/client complexes and enable a detailed dissection of the role that dynamics play in dictating the CHIP/Hsp70 triage mechanism. SAXS and EPR will be used together in a hybrid methods approach to determine structural ensembles for dynamic CHIP/Hsp70 complexes and connect dynamic conformational changes to alterations in CHIP-mediated ubiquitination of Hsp70 and misfolded clients. The proposed development of a SAXS/EPR hybrid methods approach to structure determination will produce an approach that will be applicable to many protein/protein interactions regulated by dynamic conformational changes. Structural data from NMR, SAXS, and EPR will be complemented with biophysical and biochemical approaches that enable real-time monitoring of ubiquitination and directly assay the competition between CHIP-mediated ubiquitination and Hsp70-mediated protein refolding. These assays will allow for rapid testing of mechanistic hypotheses generated from the NMR and SAXS/EPR structural studies. The proposed research program is set up to evolve by expanding to examine the role that clients play in the triage mechanism. These efforts will be extended to soluble protein clients with either perturbed refolding capabilities, or controlled numbers or locations of ubiquitination sites.
The need for a rapid and robust response to protein misfolding is ubiquitous in human physiology and defects in protein quality control pathways lead to a diverse range of pathologies including neurodegenerative disorders, cancers, ischemia reperfusion injury, and recessive hereditary cerebellar ataxias. Discovery of the mechanistic details that underlie triage of misfolded proteins to either refolding or degradative pathways will enhance our fundamental knowledge of protein quality control pathways and identify avenues that may be exploited for future therapeutic targeting.
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